Our long term goal is to elucidate the mechanisms of initiation and regulation of RNA polymerase II transcription. Our experimental approach is to reconstitute these processes with purified proteins from yeast, and to bring the power of combined biochemical and genetic analysis to bear on the mechanisms. Fractionation of yeast extracts during the previous project period yielded five initiation factors, termed a, b, d, e, and g, all of which are required for transcription by purified RNA polymerase II. The recent cloning of genes for the factors and expression of the proteins in yeast or E. coli should enable the culmination of our efforts during the coming project period. Specific aims of the proposed research are as follows: 1. To complete the development of a fully defined transcription system with a high efficiency of template utilization. 2. To complete the cloning of genes for factor b, to identify the subunit responsible for phosphorylation of the C-terminal repeat domain (CTD) of RNA polymerase II, to isolate and characterize a "holoenzyme" form of factor b, and to determine the essential role of factor b in transcription. 3. To resolve multisubunit complexes containing TATA-binding protein (TBP) on the basis of functional assays for basal and activated transcription, and to pursue a speculative idea about the role of TBP and factor e in duplex DNA melting by the isolation of a novel class of factor e mutant. 4. To identify the region(s) of factor e involved in functional interaction with RNA polymerase Il and in determination of the transcription start site by analysis of mutants and of chimeras between cerevisiae and pombe factor e's. 5. To identify the regions of the 54 kD subunit of factor g involved in DNA-binding and polymerase interaction by mutagenesis and construction of yeast-mammalian chimeras. 6. To determine the pattern of protein-protein interactions in an RNA polymerase II "holoenzyme" containing all initiation factors except TBP by quantitative sequential binding analysis, and to clone or identify the genes for six additional polypeptides of a second holoenzyme containing Srb proteins. 7. To identify and characterize intermediates on the transcription initiation pathway. 8. To form stable, paused elongation complexes. An assay based on adsorption of histidine-tagged RNA polymerase to Ni-agarose will be employed to reveal the components required and assess the role of factor g in complex stability. 9. To purify and characterize the previously described mediator(s) of transcriptional activation on the basis of three different functional assays. 10. To assess the physiologic significance of transcriptional activation in vitro by studies with TBP and activator protein mutants. Effects upon transcriptional activation in vitro and in vivo will be compared. 11. To clone the genes for seven as yet unidentified polypeptides in a complex with Swi and Snf proteins, and to investigate the role of the complex in transcriptional activation in vitro. 12. To resolve and reconstitute a CTD-dependent transcription system. At least two novel factors. one positive- and the other negative-acting are involved.